Nitro benzonitrile antimicrobial compositions and process



United States Patent 3,353,939 NITRO BENZONITRILE ANTIMICROBIAL CQMPOSITIONS AND PROCESS William N. Cannon, Greenwood, and Granville Bruce Kline and Dwight E. Morrison, Indianapolis, Ind., as signers to Eli Lilly and Company, Indianapolis, Ind., a corporation of Indiana No Drawing. Filed Aug. 8, 1963, Ser. No. 300,923 7 Claims. (CI. 44-75) This invention relates to a process for inhibiting the growth of bacterial and fungal microorganisms and to compositions therefor. More particularly this invention relates to the use of nitro-substituted benzonitriles as antibacterial and antifungal agents.

The antibacterial and antifungal agents employed in this invention have the following structural formula:

(JEN

wherein the nitro group may be substitued at any of the available positions on the benzene ring and wherein Y and Z may be hydrogen, lower alkoxy containing from 1 to 4 carbon atoms, lower alkyl containing from 1 to 4 carbon atoms, cyano, or nitro. Where only one nitro group is present, it is preferred that this group be in a position ortho or para to the cyano substituent. v

The compounds employed in the practice of this invention possess a broad spectrum of antibacterial and antifungal activity and are usfeul for suppressing the growth of undesirable microorganisms in a wide variety of applications. Thus for example, an inhibitory concentration of the compounds can be included as one of the components of the disinfectant solution commonly employed in the foot baths which are used around swimming pools, shower rooms and the like, to minimize the spread of bac terial and fungal infections. The compounds may also be employed in the solutions utilized in the cleaning of floors in such public areas. The antibacterial and antifungal agents of this invention can also be employed as additives in paints, in the treatment of leather to impart resistance to attack by microorganisms and in like situations where the outstanding antibacterial and antifungal properties of these agents are desired. An especially useful application of the subject compounds lies in their use as additives in hydrocarbon fuels for the suppression of growth of microorganisms therein.

The fact that undesirable microorganisms are present in petroleum products in storage has long been known. The

problems deriving from the harmful effects of bacteria and fungi in refined petroleum products, however, have assumed major importance only in very recent years. Although such microorganisms will grow and multiply in hydrocarbon fuels such as gasoline and are capable of utilizing such fuels as their source of carbon, they do so less efiiciently than with such other hydrocarbon fuels as kerosene. Since jet engine fuels resemble the latter, the problem is especially acute in this area. The scums, slimes and other residues which are formed by the microorganisms necessitate frequent and costly cleaning of the filters and strainers in the fuel systems of jet aircraft, promote corrosion, and constitute an ever present hazard in the operation and fueling of such aircraft. Furthermore, because of the high rate of fuel consumption of such aircraft and the consequent high volume of jet fuels which must be circulated through the filtering and straining systems, the problems resulting from cloggings are accentuated. Although the growth of bacteria and fungi is of special importance in jet fuels, the problems arising therefrom are not limited to such fuels but are also present in other types of hydrocarbon fuels such as diesel fuels, heating oils, and the like.

It has generally been thought that the presence of water is essential for the growth of such microorganisms in hydrocarbon fuels. Accordingly, methods of controlling the problem have been proposed which depend upon the careful drying of such fuels to remove substantially all of the water present therein. Apart from the expense involved in such treatment, this result is, as a practical matter, almost impossible to achieve because of the constant accumulation of water in storage tanks as the result of condensation of moisture therein and ground water seepage. Furthermore, recent evidence indicates that the microorganisms can persistrin a more or less quiescent state for a period of six months or more in fuels containing not more than 0.01 percent of water and that a veritable population explosion occurs when such fuel is finally brought into contact with water. It is thus apparent that other means of controlling the problem must be sought.

It is an object of this invention to provide a process for inhibiting the growth of bacterial and fungal microorganisms. A further object of this invention is to provide hydrocarbon fuel compositions containing additives which prevent the growth of undesirable bacterial and fungal microorganisms. Another object is to provide such compositions which do not produce corrosive by-products upon combustion. Still another object of the invention is to provide a process for inhibiting the growth of such microorganisms in hydrocarbon fuels. Other objects will become apparent to those skilled in the art from the ensuing description.

It has now been found that the outstanding antibacterial and antifungal properties of the compounds employed in this invention provide an excellent means for controlling the growth of objectionable mold, bacteria, and fungi in hydrocarbon fuels and that the difiiculties arising from the presence of scums and other residues produced by the microorganisms are circumvented. In general, the hydrocarbon fuel is in contact with an aqueous phase, this being the state in which such fuels are usually encountered under normal storage conditions.

As is apparent from the structural formula shown above, the antibacterial and antifungal agents employed in the practice of this invention include, in addition to the simple nitrobenzonitriles, compounds containing a plurality of nitro and cyano groups, and, in fact, a highly preferred compound is 2,4-dinitrobenzonitrile. Other compounds which are used effectively to inhibit bacteria and fungi include 2,3-dinitrobenzonitrile, 2-nitro-4-methoxybenzonitrile, 2-nitro-3-rnethylbenzonitrile, 2-nitro-6-methoxybenzonitrile, 2,6-dinit-robenzonitrile, 3,5-dinitrobenzonitrile, 2,S-dinitrobenzontittrile, 3,4-dinitrobenzonitrile, Z-nitrobenzonitrile, 3-nitrobenzonitrile, 4-nitrobenzontrile, Z-nitrophthalonitrile, 2-nitroterephthalonitrile, 2-nitro-4- butoxybenzonitrile, 2-nitro-4-t-butylbenzonitrile, and the like.

An illustration of the broad spectrum of activity and high degree of efficacy exhibited by the nitrobenzonitriles employed in this invention is provided in Table I, where in the minimum inhibitory concentrations of 2,4-dinitrobenzonitrile against a number of bacterial and fungal animal pathogens and saprophytes are shown. The activity of the same compound against bacterial and fungal plant pathogens and material degrading organisms is 7 shown in Table II.

TABLE I.IN VIIRO ACTIVITY OF 2,4-DINITROBENZONI- TRILE AGAINST BACTERIA AND FUNGI Minimum Inhibitory Concentration 1 Microorganism 24 hr. 48 hr.

1. 56 1. 56 Staphylococcus aureus 3055 0. 2 0. 4 Bacillus subtilz's 0. 78 1. 56 Mycobactarium aviwm 50 50 Streptococcus faecalis 1. 56 6. 25 Lactobacillus cam 50 100 Leuconostoc citrovorum 12. 5 25 Escherichia 0011' No. 1

6. 25 6. 25 Escherichia coli N o. 2

3.13 6. 25 Proteus sp. N0. 1

6. 25 12. 5 Proteus sp. No. 2 50 50 Pseudomonas sp. No. 2 25 25 Pseudomonas sp. No. 5

6. 25 12. 5 Klebsiclla-Acrbacler N0. 14 12. 25 Klebsiella-Aerobacter No. 15 12. 5 12. 5 Salmonella sp. No. 1

3. l3 6. Vibrio metschnikooii 1. 56 3.13 Saccharomyces pastoriamls 25 25 Candida albz'cans 1. 56 3. 13 Trichophyton mc'ntagrophytes 1 Meg/ml, or parts per million.

nitriles are extremely effective against a representative group of microorganisms isolated from contaminated IP-4 jet engine fuel. Among the bacterial and fungal organisms isolated therefrom are two species of Bacillus, Pseudomonas acluginosal, a green fungus tentatively identified as a species of Penicillium, a brown fungus thought to be a species of Hormodendrum, Cladiosporum resinae, a black fungus tentatively classified as a species of Aspergillus, and a gray fungus and a yellow-green fungus both of which remain unidentified. All of these organisms are controlled to a highly effective degree by the compounds of this invention, as is apparent from the follow ing description of the method used in determining the activity of the subject compounds by an agar plate procedure.

Serial dilutions of the nitrobenzonitriles were prepared in molten Penn-Assay medium and plates were poured at concentrations ranging from 12.5 to 200 ppm. After the plates h-ad hardened, they were surface-inoculated with 0.1 ml. of inoculum. For the bacterial isolates the inoculum consisted of a three-day culture growing in nutrient broth. For the fungal isolates the inoculum consisted TABLE III.MINIMUM INHIBITOR CONCENTRATIONS OF NITROBENZONITRILES AGAINST FUEL CONTAMINANTS Microorganism 2 Compound R-3 R-ll R-12 R-l 11-4 R-5 R-G R-7 C.r.

Z-nitroterephthalonitrile. 50 200 100 12. 5 l2. 5 50 12. 5 12. 5 2,5dinitrobenzonitrile... 25 25 50 200 50 50 200 25 50 2-nitro-6-methoxybenzonit 200 100 200 25 50 50 50 25 25 3,4denitr0benzonit1ile. 25 12. 5 100 100 50 12. 5 50 25 100 2,5-dinitrobenzonitrile. 12. 5 12. 5 50 50 25 12. 5 25 25 25 2-nitro-5-methoxybenzonit 200 100 200 25 50 50 50 25 25 2,4-dinitrob enzonitrile 12. 5 12. 5 50 50 25 25 50 12. 5 12. 5 2nitro-4-methylbenzonitrile 12. 5 100 200 25 200 200 100 I 100 25 3,5-dinitrobenzonitrile... 12. 5 12. 5 200 25 100 100 50 50 25 2-nitrobenzonitrile 50 100 200 100 100 50 50 50 25 2-nitro-4-methoxybenzom ri e. 50 50 200 12. 5 50 100 5 25 25 3-nitrophthalonitrile 50 50 200 200 25 100 200 12. 5 100 4-nitrophthalonitrile 100 200 200 50 12. 5 100 50 25 50 1 Meg/ml.

2 Identity of microorganisms as follows: R-3, Bacillus sp.; R-ll, Bacillus sp.; R-12, Pseudom Hormodendtum sp.; R-5, Grey fungus, unidentified; resmac.

TABLE II.ACTIVITY OF 2,4-DINITROBENZONITRILE AGAINST PLANT PATHOGENS AND MATERIAL DE- GRADING ORGANISMS Minimum inhibitory concentration 1 72 hr.: Microorganism 3.13 Agmba'cterium tumefaciens. 3.13 Erwinia amylovo ra. 0.4 Pseudomonas solana ccarum. 6.25 Xanthovno nas phaseoli. 0.78 Alternaria solani. 25 Aspergillus niger. 3.13 Botrytis cinerea.

12.5 Ceratostomella ulmi. 12.5 Co-llectotrichum pisi. 12.5 F zrsarium oxysporz'zrm f. lycopersici. 12.5 Glomerella cingulata. 3.13 Helminthosporiwm sativum. 1.56 Polyporus ostreatus. 25 Penicillium expansum. 12.5 Pullularia sp. 3.13 Sclerotinia fructicolw. 12.5 Verticillium albo atrum. 12.5 Mucor ramannianws. 6.25 Spicaria divwricata.

1 Meg/n11.

of a five-day culture growing in Sabouraud liquid medium. The plates inoculated with bacterial cultures were incubated at 37 C. for 48 hours; those inoculated with fungal cultures were incubated at room temperature for five days. At the end of the incubation period all plates were read and observed for growth as compared to the inoculated control plates. Table III below lists the minimum inhibitory concentrations for a series of compounds screened against microorganisms isolated from contaminated JP-4 jet fuel.

The compounds employed in this invention possess a number of outstanding advantages. Because of the enormous volumes of hydrocarbon fuels consumed, economic considerations require that any 'antimiorcbial agent employed therein be effective at relatively low concentrations and be available at a reasonable cost. The nitrobenzonitriles of this invention are effective against the microorganisms contaminating hydrocarbon fuels at concentrations as low as about 10 ppm. and are in addition very inexpensive. The partition coefficients of the compounds for fuel water mixtures are such that a favorable distribution of the compounds between the fuel and aqueous phases results. A further advantage of these compounds is that they are completely consumed in the combustion process, leaving neither solid residues nor corrosive by-products which could themselves give rise to serious problems. Still another advantage of the nitrobenzonitriles is that they remain fully effective in the presence of other additives normally employed in hydrocarbon fuels. The said additives include organo-metallic de- (mas aerugi'hosa; R-l, probably Penlclllium sp.; R-4, R6, yellow-green fungus, unidentified; R-7, probably Aspergillus sp.; C.r., Cladi'osporum rivatives such as organolead and organoaluminum compounds, ignition control compounds such as boron and phosphorus derivatives, deicing compositions, and the like.

In general, it is preferred to employ compounds having at least one nitro group in a position ortho to the cyano substituent. The activity of compounds with a para nitro group is frequently enhanced by the presence of further substitution in the ring. Compounds having a single nitro group in the meta position require considerably higher concentrations for efficacy, but the addition of a second nitro group greatly enhances activity, and 3,5-dinitr0- benzonitrile is one of the highly preferred compounds. An especially preferred compound is 2,4-dinitrobenzonitrile.

The compounds are employed for the control of bacteria and fungi by applying them to the locus of the microorganisms. In the case of hydrocarbon fuels, the compounds are generally added in the form of a concentrate in a combustible solvent compatible with the hydrocarbon fuel in an amount such that the final concentration is between about p.p.m. and about 1000 ppm, preferably between about 10 ppm. and about 200 ppm. The low cost of the subject compounds renders use at these concentrations eminently practical.

The practice of the invention will be better understood in the light of the illustrative examples which follow.

Example 1 Solutions of 2,5-dinitrobenzonitrile in JP4 jet fuel which have been sterilized 'by Seitz filtration are prepared providing concentrations between 62.5 and 1000 ppm. Three hundred milliliter portions of the solutions are placed over ml. of an aqueous base consisting of Bushnell-Haas medium a simple mineral salts medium which contains no source of carbon. The fuel-medium mixtures are placed in 250 ml. of flasks which are sterilized prior to use. Each of the flasks containing the fuel-water mixture is inoculated with 0.3 ml. of an inoculum containing 675x10 organisms per ml. which is a blend of both bacterial and fungal cultures containing organisms normally found as contaminants in this type of jet fuel. A number of flasks containing the fuel-medium mixture without additives are also inoculated to serve as controls. At weekly intervals a sterile pipette is employed to withdraw a small volume of the aqueous phase. This is streaked onto a nutrient agar plate which is then incubated and observed for signs of growth of the microorganisms. The plates are incubated at 37 C. for 48 hours to detect bacterial growth, while the plates employed to detect fungal growth are kept at room temperature for five to seven days. The absence of growth on the plates at the end of the incubation period reflects an absence of microbiological contamination in the fuelaqueous system. None of the samples withdrawn ,at the end of one, two, three or four weeks produce any detectable growth on the plates inoculated therewith at any of the concentrations. Inoculated controls containing no additive are all positive at each of the Weekly readings.

Example 2 The general procedure of Example l is followed except that concentrations of 125, 250, 500 and 1000 ppm. of 2,4-dinitrobenzonitrile are employed. The additive is provided by adding to the fuel mixture a suitable quantity of a concentrate comprising a 10 percent solution of 2,4-dinitrobenzonitrile in methyl Cellosolve to which a small quantity of glycerine has been added. In addition, instead of determining the presence of microorganisms by observing growth on seeded plates, actual plate counts are done in order to determine the number of organisms present in the aqueous phase at various time intervals. The mixed inoculum again contains 675 10 organisms per ml. All of the flasks containing the 2,4-dinitrobenzonitrile are entirely free of bacterial and fungal organisms at each observation period. The number of microorganisms per milliliter present in the untreated control flask, which also contained methyl Cellosolve and glycerine, are as follows: 74 10 after three days, 52 l0 after one week, and 116 10 after two weeks.

A similar procedure employing lower concentrations of 2,4-dinitrobenzonitrile discloses that the mixture remains free of microorganisms at concentrations of 2,4- dinitrobenzonitrile of 30 ppm. and above.

Example 3 The procedure of Example 2 is repeated using Z-nitrobenzonitrile as the additive. All flasks containing 250 p.p.m. or more of the additive are entirely free of bacterial or fungal contamination at each observation. The control flasks contain organisms as indicated in Example 2.

Example 4 The procedure of Example 2 is repeated with identical results using 3,4-dinitrobenzonitrile. In this case the additive is provided as a concentrate in benzene solution.

We claim:

1. A hydrocarbon fuel composition comprising essentially a major portion of a hydrocarbon fuel and an inhibitory concentration of .an antimicrobial compound selected from the group consisting of 2-nitrobenzonitrile. 2,4-dinitrobenzonitrile, 2-nitroterephthalonitrile, 3-nitrophthalonitrile, and 4-nitrophthalonitrile.

2. The hydrocarbon fuel composition of claim 1 wherein the said antimicrobial compound is present in a concentration between about 10 and about 1000 ppm.

3. The composition of claim 1 in which the antimicrobial compound is 2-nitrobenz-onitrile.

4. The composition of claim 1 in which the antimicrobial compound is 2,4-dinitrobenzonitrile.

5. The composition of claim 1 in which the antimicrobial compound is 2-nitroterephthalonitrile.

6. A composition for the prevention of fouling of hydrocarbon fuels which comprises a concentrate of an antimicrobial compound selected from the group consisting of Z-nitrobenzonitrile, 2,4-dinitrobenzonitrile, 2-nitroterephthalonitrile, 3-nitrophthalonitrile, and 4-nitrophthalonitrile in a combustible solvent which is compatible with the hydrocarbon fuel.

7. The composition of claim 6 in which the antimicrobial compound is 2,4-dinitrobenzonitrile.

References Cited UNITED STATES PATENTS 2,975,042 3/1961 Summers 44-72 2,975,043 3/1961 Ambrose 4472 OTHER REFERENCES Koopmans (II) Medel: Landbounhage School Opzoekingssta. Staat Gent, 1962, vol. 27(3), pp. 1204-1213, (Chemical Abstracts, 1964, vol. 60, p. 1579(e)). Photocopy can be found in Group 120-260/465 G UXR.

DANIEL E. WYMAN, Primary Examiner.

Y. M. SMITH, Assistant Examiner. 

1. A HYDROCARBON FUEL COMPOSITION COMPRISING ESSENTIALLY A MAJOR PORTION OF A HYDROCARBON FUEL AND AN INHIBITORY CONCENTRATION OF AN ANTIMICROBIAL COMPOUND SELECTED FROM THE GROUP CONSISTING OF 2-NITROBENZONITRILE, 2,4-DINITROBENZONITRILE, 2-NITROTEREPHTHALONITRILE, 3-NITROPHTHALONITRILE, AND 4-NITROPHTHALONITRILE. 